Identification and characdterization of DNA glycosylases that recognize and remove oxidative base damage in DNA

(1)活性氧物种引起DNA中的嘌呤和嘧啶的各种氧化修饰。细菌和真核生物已经进化出DNA氧化碱基损伤的碱基切除修复机制。大肠杆菌有三种DNA糖基酶，MutM，Nth和Nei，它们识别和去除DNA中氧化损伤的碱基。这些DNA糖基酶能够识别和去除DNA中的5-甲酰尿嘧啶和5-羟甲基尿嘧啶。然而，一些证据表明，E.coliMnth nei三重突变体的粗提物中仍有残留活性。在本研究中，我们鉴定了新型酶(S)识别含有5-FOU的双链寡核苷酸的能力。(2)7，8-二氢-8-氧鸟嘌呤(8-oxoG)是DNA中最重要的氧化碱基损伤产物，在细菌和哺乳动物细胞中引起G：C到T：A的转换。8-oxoG在大肠杆菌中被MutM修复，在酵母和哺乳动物细胞中被8-oxoG-DNA糖基酶(Ogg1)修复。在本研究中，我们鉴定了…更多，并鉴定了人的hOgg1的海鞘同源物在肠黄瓜(CiOgg1)。CiOgg1基因的导入显著降低了自发性G：C到T：A颠换的频率。纯化的GST-CiOgg1融合蛋白具有8-oxoG DNA糖基酶/AP裂解酶活性。它与含8-oxoG的双链寡核苷酸形成席夫碱中间体，并优先从8-oxoG/C上脱除8-oxoG。CiOgg1通过β消除反应裂解含8-oxoG的双链DNA。此外，还比较了CiOgg1在不同组织中的表达水平。在睾丸中的表达水平最高。(3)对黄瓜AP内切酶(CiAPE)进行了纯化和鉴定。比较了CiAPE和人APE1的性质。CiAPE蛋白大小约为34 kDa。它能有效地切割含四氢呋喃(THF)的双链寡核苷酸。此外，CAPE对大肠杆菌BW9093ΔXth的转化增强了对H_2O_2的敏感性。这些结果表明，CiAPE具有AP内切酶活性，可以防止黄瓜体内的氧化应激反应。较少

(1) Reactive oxygen species cause a wide variety of oxidative modifications to purines and pyrimidines in DNA. Bacteria and eukaryotes have evolved base excision repair mechanisms for oxidative base damage in DNA. E.coli has three kinds of DNA glycosylase, MutM, Nth and Nei, that recognize and remove oxidatively damaged bases from DNA. These DNA glycosylases are able to recognize and remove 5-formyluracil and 5-hydroxymethyluracil in DNA. However, several evidence showed that there are residual activities in crude extract from E.coli mutM nth nei triple mutant. In this study, we identified the abilities of novel enzyme(s) to recognize double-stranded oligonucleotides containing 5-foU. (2) 7,8-dihydro-8-oxoguanine (8-oxoG) is the most important product of oxidative base damage in DNA, and causes G:C to T:A transversions in bacteria and mammalian cells. 8-oxoG is repaired by MutM in E.coli and 8-oxoG-DNA glycosylase (Ogg1) in yeast and mammalian cells. In the present study, we identified … More and characterized an ascidian homolog of the human hOgg1 in Ciona intestinalis (CiOgg1). Introduction of the CiOgg1 gene significantly reduced the frequency of spontaneous G:C to T:A transversions in E.coli mutM mutY strain. Purified GST-CiOgg1 fusion protein had 8-oxoG DNA glycosylase/AP lyase activity. It formed Schiff base intermediates with 8-oxoG-containing duplex oligonucleotides and removed 8-oxoG preferentially from 8-oxoG/C. The CiOgg1 cleaved 8-oxoG-containing duplex DNA via β-elimination reaction. Furthermore, the expression level of CiOgg1 was compared in various tissues in Ciona intestinalis. The highest expression level was observed in testis. (3) In this study, AP endonuclease in Ciona inteslinalis (CiAPE) was purified and characterized. The properties of CiAPE were compared with those of human APE1. The CiAPE protein was about 34 kDa. It efficiently cleaved tetrahydrofuran (THF)-containing duplex oligonucleotides. In addition, the transformation of E.coli, BW9093 Δxth with CiAPE complemented the H2O2 sensitivity. These results demonstrated that CiAPE has AP endonuclease activity to prevent against oxidative stress in Ciona intestinalis. Less